The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor nucleic acid sequences (Gusella, Ann. Rev. Biochem., 55:831-854 (1986). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form, or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.
Several different types of polymorphism have been reported. A restriction fragment length polymorphism (RFLP) is a variation in DNA sequence that alters the length of a restriction fragment. The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses. When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.
Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetra-nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis, and in a large number of genetic mapping studies.
Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs. Some single nucleotide polymorphisms (SNPs) occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case, one of the polymorphic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease. Examples of genes in which polymorphisms within coding sequences give rise to genetic disease include: globin (sickle cell anemia), apoE4 (Alzheimer's Disease), Factor V Leiden (thrombosis), and CFTR (cystic fibrosis). cSNPs can alter the codon sequence of the gene and therefore specify an alternative amino acid. Such changes are called “missense” when another amino acid is substituted, and “nonsense” when the alternative codon specifies a stop signal in protein translation. When the cSNP does not alter the amino acid specified the cSNP is called “silent”.
Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects. Single nucleotide polymorphisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages.
Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. The different forms of characterized single nucleotide polymorphisms are often easier to distinguish than other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers).
Only a small percentage of the total repository of polymorphisms in humans and other organisms has been identified. The limited number of polymorphisms identified to date is due to the large amount of work required for their detection by conventional methods. For example, a conventional approach to identifying polymorphisms might be to sequence the same stretch of DNA in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.
The renin-angiotensin-aldosterone system (RAAS) represents an endocrine system that plays a fundamental role in cardiovascular function by regulating extracellular fluid volume and sodium balance. Activation of RAAS is triggered with the release of renin from the kidney, resulting in proteolytic cleavage of angiotensinogen to angiotensin I (AT1). AT1 is converted to angiotensin II (AT2) by angiotensin-converting enzyme (ACE). Additional non-renin and non-ACE-dependent mechanisms also exist for the production of AT2 directly from angiotensinogen and from AT1. Binding of AT2 to the angiotensin II receptor type 1 (AT2R1) stimulates the production of the steroid hormone aldosterone which mediates its effects through the mineralocorticoid receptor (MLR), promoting renal sodium retention and potassium loss.
Dysregulation of RAAS is associated with a number of disease states including renal injury and most forms of hypertension. Modulation of RAAS has been achieved clinically with compounds that suppress AT2 and aldosterone production (e.g. ACE inhibitors, AT2R1 blockers) as well as compounds that suppress aldosterone action (e.g. MLR antagonists).
Thiazolidinediones (TZDs) are high affinity PPARγ ligands which induce the expression of genes involved in glucose homeostasis and insulin sensitivity. PPARγ is expressed primarily in adipose tissue, where much of its antidiabetic actions are believed to be manifested. However PPARγ plays a significant role in vascular tissues as well. Expression of PPARγ has been detected in endothelial cells as well as vascular smooth muscle cells. PPARγ also plays a role in macrophages where its induction results in anti-inflammatory responses via inhibition of iNOS, MMP9 and scavenger receptor A genes. PPARγ has also been shown to play significant anti-inflammatory roles in monocytes and in atherosclerotic plaques.
An observed, adverse event associated with PPARγ-agonist treatment is the development of dose-dependent peripheral edema. Although the underlying mechanism is currently unknown, PPAR-agonist induced edema is believed to arise through direct action on the kidney (Hollenberg N K, Am J Med 115(8A):111-115 (2003)). Several lines of evidence have implicated a role for PPARγ agonists in the modulation of RAAS. In vascular smooth muscle cells, treatment with PPARγ agonists was shown to antagonize the transcription of the AT2R1 receptor, resulting in both reduced AT2R1 mRNA and protein levels (Sugawara et al., Endocrinology 142:3125-3134 (2000); Takeda et al., Circulation 102:1834-1839 (2000)). Moreover, animal model studies have shown that treatment with TZDs, including pioglitazone and rosiglitazone, mitigated hypertension in AT2-infused rats, in part, via inhibition of AT2R1 and induction of angiotensin II receptor type 2 (Diep et al., Circulation 105:2296-2302 (2002)). As RAAS plays a fundamental role in fluid volume homeostasis, it is conceivable that modulation of other components of this system, by PPAR agonists, might contribute to agonist-induced edema mechanism of action.
The endothelins (ETs) constitute a family of three separate genes (ET-1, ET-2, ET-3) that encode tissue-specific precursor peptide products (Gianessi et al., 2001). All three ET isoforms are proteolytically processed to generate 21 amino acid vasoactive peptides. ET-1 is the predominant isoform that is secreted by vascular endothelial cells in response to endothelial cell activators such as thrombin, TNF-α and angiotensin II (Gianessi et al., 2001; Delerive et al., 1999).
Endothelin-1 (ET-1) plays a role in a number of biological processes including monocytic chemotaxis, induction of endothelial cell adhesion molecules as well as vascular tone maintenance (Gianessi et al., 2001; Delerive et al., 1999). ET-1 is the most potent vasoconstrictor known and, as such, plays a important role in blood pressure elevation in several animal models of hypertension (Schiffrin, 2001). ET-1 also participates in salt and water homeostasis via cross-talk with the Renin-Angiotensin-Aldosterone-System (RAAS) (Agapitov and Haynes (2002)
Elevated ET-1 secretion is associated with a number of clinical states including hypertension, atherosclerosis, heart failure and renal failure (Luscher and Barton, 2000). Hyperinsulinemia and/or insulin resistance is also associated with increased ET-1 secretion from vascular endothelial cells in vitro and in vivo (Satoh et al., 1999). ET-1 receptor antagonists possess hypotensive effects in animal models of hypertension (Luscher and Barton, 2000) and in patients with essential hypertension (Satoh et al., 1999). Taken together, these observations suggest that elevated ET-1 may play a role in type 2 diabetes-associated hypertension (Satoh et al., 1999).
Recent studies have shown that ET-1 secretion is suppressed by PPARα and -γ agonists in endothelial and vascular smooth muscle cells (Fukunaga et al., 2001; Delerive et al., 1999; Satoh et al., 1999). The underlying mechanism of action is through negative interference of PPARα and -γ with the Activator Protein-1 signaling pathway (Delerive et al., 1999), which regulates ET-1 transcriptional activity (Kawana et al., 1995). This inhibition may contribute to the hypotensive effect observed with PPAR-agonist treatment in diabetic patients (Satoh et al., 1999).
The roles described above for ET-1 in vascular tone maintenance, along with its involvement in numerous cardiovascular disease processes suggest ET-1 may potential be involved in PPAR-agonist induced edema mechanism of action.
Genetic polymorphisms in members of the renin-angiotensin-aldosterone system, in addition to other proteins described herein, may cause alterations in the level of renin or its related peptides, or may affect downstream signal transduction. Genetic polymorphisms in ET-1 may also cause alterations in the level of ET-1 or its related peptides, or may affect downstream signal transduction. Such polymorphisms may genetically predispose certain individuals to an increased risk of developing edema, particularly in response to PPAR-agonist induced therapy. Such polymorphisms are expected to show a significant difference in allele frequency between individuals treated with a high dose of a PPAR-agonist who do not exhibit edema and individuals treated with a PPAR-agonist who do exhibit edema. Genotypes of such polymorphisms can predict each individual's susceptibility to edema, and thus will be useful in identifying a group of high risk individuals that may be subject to modified PPAR-directed treatment regimens. Alternatively, the identification of such a group may preclude one or more individuals within said group from being administered an PPAR-directed agonist or antagonist.
Moreover, the present invention also provides, for the first time, strong supporting evidence that renin expression is specifically upregulated and endothelin-1 expression is specifically downregulated in response to PPAR-agonist treatment and thus serves as an effective predictive marker for PPAR-agonist efficacy in addition to aiding in the identification of individuals at risk of developing PPAR-agonist induced dose-dependent peripheral edema.
The β-adrenergic receptor family consists of three genes (β1, β2, β3) that encode membrane-bound G-protein coupled receptors (Dzimiri et al., 1999). All three receptor subtypes respond to extracellular signals by increasing intracellular cAMP levels via G-protein dependent adenylyl cyclase activation. Increased cAMP levels activate cAMP-dependent protein kinase A (PKA), which, in turn, phosphorylates a variety of target proteins to elicit cellular responses (Castellano and Bohm, 1997; Dzimiri et al., 1999)
In cardiac tissue, β-adrenergic signaling enhances overall cardiac output by increasing heart rate and contractility in response to catecholamine-induced receptor stimulation. These effects are brought about by PKA-dependent phosphorylation of target genes that are essential for cardiac function including L-type calcium channels, phospholambin-a, troponin I and myosin binding protein C. (Lohse et al., 2003; Bengtsson et al., 2001; Dzimiri, 1999). Together, these target proteins mediate their effects through the regulation of calcium activity (e.g. availability and sensitivity) in cardiomyocytes. Although both β1 and β2 contribute to these cardiac effects, most of the functional effects are mediated through β1 subtype (Lohse et al., 2003) β1 also mediates other cellular responses relevant to cardiac function including transient relaxation of blood vessels (Chruscinski et al., 2001) and activation of RAAS by enhancing renin secretion in the kidneys (Dzimiri et al., 1999).
The roles described above for β1-adrenergic receptor in regulating cardiac performance directly within cardiomyocytes as well as indirectly via actions on kidney function suggest that the β1-adrenergic receptor may be associated with the mechanism of action of PPAR-agonist induced edema.
Genetic polymorphisms in the β1-adrenergic receptor may cause alterations in the level of the β1-adrenergic receptor or its related peptides, or may affect downstream signal transduction. Such polymorphisms may genetically predispose certain individuals to an increased risk of developing edema, particularly in response to PPAR-agonist induced therapy. Such polymorphisms are expected to show a significant difference in allele frequency between healthy individuals and edema subjects. Genotypes of such polymorphisms can predict each individual's susceptibility to edema, and thus will be useful in identifying a group of high risk individuals that may be subject to modified PPAR-directed treatment regimens. Alternatively, the identification of such a group may preclude one or more individuals within said group from being administered an PPAR-directed agonist or antagonist.